Since Szentivanyi proposed in 1968 that asthma might be due to an inherited or acquired deficit in beta-adrenergic function, much research has focused on the beta-adrenergic receptor (beta2AR) and the beta- adrenergic cascade. However, 22 years later the question is still unsettled, as research has focused largely on the leukocyte, which is poor model of human asthma. Studies of bronchial muscle tissue from animal models of asthma and from human asthmatics obtained either at surgery or at autopsy show a very definite deficit in beta-adrenergic function. However, the intracellular events underlying this beta- adrenergic deficit are unknown. We have a well characterized animal model of airway hyperresponsiveness that clearly demonstrates decreased sensitivity to beta-adrenergic agonists in vivo and in vitro. We hypothesize that the decreases sensitivity to beta-adrenergic agonists in vivo and in vitro. We hypothesize that the decreased sensitivity to beta-adrenergic agonists relates to a specific defect either in generation or degradation of intracellular cAMP. To identify directly the intracellular lesion we plan to measure numbers and affinities of beta2AR and muscarinic receptor subtypes, second messengers, adenylyl cyclase and their regulation by the signal transducing G proteins in the airway smooth muscle of the basenji-greyhound dog. We are currently measuring adenylyl cyclase activity in the white blood cell and airway smooth muscle. We have identified the presence of Gs/alpha, Gi/alpha, and beta gamma using human polyclonal antibodies in Western blots. Preliminary studies in the white blood cell reveals decreased adenylyl cyclase activity in response to beta agonists in the BG dog despite normal quantities of G proteins. The reduced in vitro sensitivity to beta agonists in our animal model provides us with the unique opportunity of easy accessibility to large quantities of airway smooth muscle for our in vitro studies from tho allergic lines of animals - one hyperresponsive and one normoresponsive, allowing us to focus on the deficit leading to hyperresponsiveness, not confounded by allergy or drug therapy. We also have the unique ability to control and manipulate environmental and genetic influences. Results from the proposed work will provide new information about the intracellular mechanisms which regulate airway smooth muscle tone in this model.